1. Technical Field
This invention relates to human cytomegalovirus (HCMV), and in particular to peptide fragments from a protein that produces T-cell epitopes of HCMV in human beings. The peptide fragment epitopes are capable of directing human cytotoxic T lymphocytes (CTL) to recognize and lyse human cells infected with HCMV.
2. Description of the Background Art
The HCMV genome is relatively large (about 235,000 base pairs) and can encode more than two hundred proteins. HCMV comprises a nuclear complex of double-stranded DNA surrounded by capsid proteins having structural or enzymatic functions, and an external glycopeptide- and glycolipid-containing membrane envelope.
HCMV infection is relatively common and is usually self-limiting in the healthy, immunocompetent child or adult (L. Rasmussen, Curr. Top. Microbiol. Immunol. 154:221-254 (1990)). However, the virus can cause severe disease in the fetus or infant. For example, HCMV is a common cause of congenital mental retardation in children who acquire the infection in utero from mothers carrying an active infection. Other newborn infants can carry cytomegalovirus for some time before they show symptoms of the disease. Approximately 10% of all newborn infants carry HCMV.
Patients with an active HCMV infection often suffer impairment of at least some of their vital organs, including salivary glands, brain, kidney, liver and lungs. Furthermore, HCMV is associated with a wide spectrum of classical syndromes including mononucleosis and interstitial pneumonia. HCMV also has an oncogenic potential and a possible association with certain types of malignancies, including Kaposi""s sarcoma.
Persistent and apparently asymptomatic HCMV infection in an otherwise healthy adult also may pose health risks in certain individuals. For example, individuals who have undergone coronary angioplasty sometimes subsequently develop restenosis as a result of arterial remodeling. In one study, about one third of such patients with restenosis had detectable HCMV DNA in their arterial lesions (E. Speir et al., Science 265:391-394 (1994)), whereas in another study HCMV seropositive patients were five times more likely to develop restenosis than their seronegative counterparts (Y. F. Zhou et al., New England J. Med. 335:624-630 (1996)). These studies suggest that decreasing the number of HCMV infected host cells can benefit certain individuals.
HCMV also has been associated with morbidity and mortality in immunocompromised patients. HCMV is an important consideration in the treatment of patients suffering from Acquired Immunodeficiency Syndrome (AIDS). The defining complication of HCMV is viral retinitis, which, if left untreated, can lead to blindness. Other disease manifestations of HCMV viremia include encephalitis, enteritis and pneumonia. At autopsy there is multi-organ involvement of HCMV disease in the majority of AIDS patients who had severe HCMV retinitis. Historically, HCMV disease has been one of the more devastating of the opportunistic infections that beset HIV-infected individuals whose CD4+ T cell level diminishes below 100/mm3.
HCMV can cause opportunistic infections in, for example, immunosuppressed organ transplant patients. Prior to the use of antiviral chemotherapy, HCMV infection had been responsible for a substantial proportion of post-bone marrow transplantation complications (J. Meyers et al., J. Infect Dis. 153:478-488 (1986)). The use of drugs such as gancyclovir with substantial anti-HCMV activity have reduced complications associated with post-bone marrow transplant HCMV infections (G. Schmidt et al. New England J. Med. 324:1005-1011 (1991); J. M. Goodrich et al., New England J. Med. 325:1601-1607 (1991)). However, prophylactic administration of gancyclovir has several negative consequences, including neutropenia and increased numbers of fatal bacterial and fungal diseases. Equally importantly, gancyclovir also delays reconstitution of cellular immunity as well as specific cellular responses to CMV. This results in a complication referred to as xe2x80x9clate CMV disease,xe2x80x9d which arises about 90 days post-transplant. Late CMV disease can result in morbidity or mortality and is most common in patients who have received either prophylactic or therapeutic gancyclovir treatment soon after transplant.
A CD8+ CTL response is believed to be important in a mammalian host response to acute viral infections such as HCMV. The observations that HCMV infection is widespread and persistent, and may be reactivated and become clinically evident in the immunosuppressed patient, suggest that virus-specific T-cells play an important role in the control of persistent infection and the recovery from HCMV disease.
In humans, protection from the development of HCMV disease in immunosuppressed bone marrow transplant recipients correlates with the recovery of measurable CD8+ HCMV-specific class I MHC-restricted T cell responses (Quinnan et al., N. Eng. J. Med. 307:7-13 (1982); Reusser et al., Blood 78:1373-1380 (1991)). The transfer of donor-derived HCMV-specific CD8+ CTL clones to allergenic bone marrow transplant recipients results in detectable CTL-based HCMV immunity, and statistically significant diminution of HCMV disease after bone marrow transplant (Walter et al., N. Eng. J. Med. 333:1038-1044 (1995)). Although successful in application, this approach has the disadvantage that it requires a sophisticated laboratory setup, which is also highly labor-intensive and costly, to derive the HCMV-specific CTL in vitro.
Because human cytomegalovirus is relatively common, yet is associated with some extremely serious health conditions, a vaccine which can reduce disease incidence and severity in a bone marrow transplant recipient, a solid organ transplant, a heart patient, an AIDS patient or a woman of child-bearing years would be highly desirable. Several HCMV vaccines are in development, including live attenuated CMV, CMV proteins carried in attenuated pox-viruses and soluble analogs of CMV membrane proteins. Unfortunately, the FDA has not approved any of these vaccines as safe and effective, despite the great efforts made in their development.
Vaccine development using CTL epitopes has become a widely adapted strategy to immunize individuals against infectious diseases and cancer. The specificity of CTL epitopes, and the fact that intracellular protein processing is not required, makes them an attractive alternative to the use of whole proteins as immunogens. To develop such a vaccine, the viral proteins which cause the host to recognize HCMV must be identified.
A variety of antigens, including tumor antigens, viral antigens and self-proteins are processed into peptides which are delivered to MHC Class I for presentation on the surface of antigen presenting cells (Reddehase et al., Nature 337:651-653 (1989); Rosenberg et al., Nat. Med. 4:321-327 (1998); Visseren et al., J. Immunol. 154:3991-3998 (1995)). Since the discovery that 8-12 amino acid fragments of cellular or viral proteins are embedded in the peptide binding groove of MHC Class I, there has been considerable interest in identifying the amino acid sequence of these fragments (Joyce and Nathenson 1994; Rammensee et al. 1993). Some of these peptides have been identified, formulated into vaccines, and evaluated for efficacy against certain viral diseases and cancer (Vitiello et al. 1995; Wang et al. 1990).
The viral life cycle provides insight as to the most effective time frame for targeting a vaccine to maximally disrupt virus production and spread. Following HCMV entry into the host cell and uncoating, the viral genome is expressed sequentially via immediate early (0-2 hour), early (2-24 hour) and late ( greater than 24 hour) viral proteins. However, certain viral structural proteins such as pp65 and pp150 are chaperoned into the cell because of their existence in large quantity in the viral particle.
The viral structural protein, pp150, has been identified as a target antigen for HCMV-specific class I MHC restricted CTL derived from the peripheral blood of most asymptomatic HCMV seropositive individuals. CTL against pp150 or pp65 (another matrix protein that is recognized frequently) are able to recognize and lyse HCMV-infected cells in vitro within an hour of infection and in the absence of viral gene expression (Riddell and Greenberg, Curr. Top. Microbiol. Immunol. 189:9-34 (1994)). Thus, CTL against HCMV pp150 are important effector cells to limit HCMV reactivation and progression to disease. The ability to induce such a cellular immune response in both immunocompromised and normal individuals would be extremely important in creating an effective vaccine (Li et al., Blood 83:1971-1979 (1994)). Peptides based on pp65 sequences which are useful for vaccines are described in U.S. Pat. No. 6,074,645, the disclosures of which are hereby incorporated by reference.
Individual MHC Class I molecules preferentially bind peptides of a given motif. The amino acid sequence of specific positions of the motif are invariant, allowing a given peptide to bind to MHC Class I molecules with high affinity. These invariant amino acids are referred to as xe2x80x9canchor positionsxe2x80x9d (Falk et al., Nature 351:290-296 (1991)). Later studies have suggested that amino acid positions other than the anchor positions also contribute to the specificity of peptide binding to MHC Class I molecules. Additionally, residues at positions within the CTL epitope which do not interact with MHC Class I molecules may interact with T cells, presumably by binding the T Cell receptor (TCR). The binding of amino acid residues to MHC or TCR structures is independently governed, so that substitution of TCR binding amino acid residues in many cases will not interfere with binding to the MHC molecule on the surface of an antigen presenting cell.
Edman degradation followed by N-terminal sequence analysis has been used to sequence the peptides which are bound to the MHC class I peptide binding groove. Mass spectrometry of HPLC-separated peptide mixtures can elucidate the primary sequence of individual peptides. In most cases, the length of these peptides is between 9 and 11 amino acids. Peptide fragments which bind to MHC are referred to as xe2x80x9cnaturally processed epitopes.xe2x80x9d
Some workers have attempted to predict which peptides of a given length, between 9-11 amino acids, will optimally bind to individual HLA Class I alleles based solely on their conformity to a motif specific for that allele. (Falk et al., Nature 351:290-296 (1991)). However, these methods do not reliably predict either correct binding or recognition by T cells as a result of endogenous processing of viral protein.
Experience with another HCMV protein, pp65, has indicated that the available motif programs are not sufficiently adept at correctly predicting sequences which are recognized by human T-cells specific for an immunogenic viral protein. Identification of naturally processed epitopes generally requires brute-force approaches, including truncation analysis, overlapping peptides, and peptide deletions consisting of single amino acid removal from either the amino or carboxyl terminus followed by assay for recognition and binding. Therefore, epitope mapping is almost completely empirical. Andersen et al., Tissue Antigens 55:519-531 (2000).
CTL are an important means by which a mammalian organism defends itself against infection by viruses and possibly cancer. A processed form of antigen, such as a viral protein minimal cytotoxic epitope, is recognized by T cells in combination with MHC Class I molecules. Functional studies of viral and tumor-specific T cells have confirmed that a minimal cytotoxic epitope consisting of a peptide of 8-12 amino acids can prime an antigen presenting cell to be lysed by CD8+ CTL, as long as the antigen presenting cell presents the epitope in the context of the correct MHC molecule.
The route of entry of a protein into the cell determines whether it will be processed as an antigen bound to either MHC Class I or Class II molecules. The endogenous or Class I pathway of protein degradation is often used by cells when infectious viruses are present. Viral nucleoproteins are processed within the cell, and degraded portions are transported to the surface via MHC Class I molecules. Viral envelope glycoproteins, because they are cell surface molecules, do not obligatorily induce CTL recognition. Viral nucleoproteins, predominantly in the form of processed epitopes, frequently are the target antigens recognized by CD8+ CTL (Townsend et al., Philos. Trans. R. Soc. Lond. (Biol). 323:527-533 (1989)).
Antigens entering the cell through exogenous pathways (pinocytosis, etc.) typically are not processed and presented by Class I MHC molecules. Methods to introduce proteins directly into the cytoplasm, therefore, have become one focus of vaccine developers. Recombinant vaccinia viruses can be used to infect cells, delivering a large amount of intracellular antigen, however these viruses themselves have the potential to cause disease in immunosuppressed people, such as bone marrow transplant recipients or AIDS patients. Attenuated vaccinia viruses, such as modified vaccinia ankara or canary pox viruses offer an alternative to immunosuppressed individuals with respect to delivery of antigens and proteins. Recent published reports have advocated the use of epitope vaccines in the minimal form, whether they are delivered as proteins made from viruses, or utilizing minimal epitope in the form of peptides. Ishioka et al., J. Immunol. 162:3915-3925 (1999); Fu, J. Virol. 72(2):1469-1481 (1998); Rodriguez et al., J. Virol. 72(6):5174-5181 (1998). Another approach to vaccination is to mix an antigenic protein with an adjuvant and introduce the mixture under the skin by subcutaneous injection.
Another potential approach to elicit cytotoxic T lymphocytes is to use the minimal cytotoxic epitope defined for a specific viral antigen in the context of a particular MHC restriction element to boost a T cell memory response to the virus. The ability of a minimal cytotoxic epitope to provide protective immunity to challenge by a lethal dose of an infectious virus has been discussed in the literature. Vaccine developers have developed increasing interest in utilizing the minimal cytotoxic epitope as the vaccine because it is capable of binding to MHC Class I molecules through external binding of the cell surface molecules without the need for internalization or processing.
Minimal cytotoxic epitopes generally have been most effective when administered in the form of a lipidated peptide together with a helper CD4 epitope (Vitiello et al., J. Clin. Invest. 95:341-349 (1995) and Livingston et al., J. Immunol. 159:1383-1392, 1997)). Peptides administered alone, however, also can be highly effective. Other vaccine modifications which have been discussed include inclusion of a signal sequence such as KDEL for endoplasmic reticulum retention and targeting to attain maximum activity. There is also evidence in the literature that a minimal cytotoxic epitope presented by particular types of antigen presenting cells (e.g. dendritic cells) may cause a primary immune response to occur in the absence of viral infection or prior contact with the virus or tumor cell.
The peptides and functional sequence variants thereof can be formulated as a vaccine as a chimeric lipidated peptide or a chimeric peptide with a covalently bound HTL epitope at the amino terminus. The HTL epitope can be any peptide that has broad reactivity to human MHC class II to stimulate a classic helper response. Such molecules include but are not limited to amino acids 830-843 from tetanus toxin (P. Panina-Bordignon et al., Eur. J. Immun. 19:2237-2242 (1989)), HTL epitopes from HIV envelope protein (J. A. Berzofsky et al., J. Clin. Invest. 88:876-884 (1991)), or a synthetic version (PADRE) predicted from known anchor residues (J. Alexander et al., Immunity 1:751-761 (1994)).
The lipidation of the HTL+CTL epitope preferably is performed on the amino terminus of the HTL epitope, with the HTL epitope being amino terminal to the CTL epitope. Suitable lipid moieties are known and described in the literature. (H. Schild et al., Eur. J. Immunol. 21:2649-2654 (1991); A. Vitiello et al., J. Clin. Invest. 95:341-349 (1995); K. Deres et al., Nature 342:561-564 (1989)). Alternatively, the CTL epitope can be lipidated at its amino terminus, followed by the HTL epitope, or the lipid can be attached at the carboxyl terminus followed by either the CTL or HTL epitope(s). Unlipidated vaccines, as well as mono-, di- and tri-lipidated vaccines are contemplated for use with the present invention. A three amino acid spacer can be inserted between the HTL and CTL epitope, or the epitopes can be fused directly in frame. Alternatively the CTL epitope lipidated on its amino terminus can be administered together with the HTL epitope, without covalent attachment.
In spite of significant efforts to identify the particular HCMV antigens and epitopes that are recognized by CTL, these naturally processed epitopes, along with effective methods of preventing and treating HCMV infection are not commercially available. Therefore, a peptide-based vaccine for this clinically important disease would be of enormous value.
Accordingly, the present invention comprises peptides according to SEQ ID NOS: 1 and 2. In a further embodiment, the invention comprises vaccines against human cytomegalovirus comprising a peptide selected from the group consisting of SEQ ID NOS: 1 and 2.
In yet a further embodiment, this invention comprises a cellular vaccine against human cytomegalovirus which comprises antigen presenting cells that present a peptide selected from the group consisting of SEQ ID NO: 1 and 2.
In yet a further embodiment, the invention comprises a recombinant viral vector which expresses a gene encoding a peptide according to SEQ ID NO: 1 or 2.
In yet a further embodiment, the invention comprises methods of modulating the immune response to human cytomegalovirus infection which comprises administering a vaccine or a cellular vaccine as described above.
In yet a further embodiment, the invention provides a method of vaccinating a mammal in need thereof against human cytomegalovirus which comprises administering to said mammal a vaccine or a cellular vaccine as described above.
In yet further embodiments, the invention provides vaccines against human cytomegalovirus which comprise a peptide selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 and an adjuvant, preferably a DNA adjuvant.
In yet a further embodiment, the invention provides an immunological reagent which comprises a peptide selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2.
The present invention is directed toward peptides which are useful for creating effective vaccines against HCMV. These peptide vaccines are able to elicit a cellular immune response against HCMV-infected cells because they are the exact epitopes which are recognized by the immune cells of persons who have been infected with HCMV and have mounted a successful response to the infection. These peptides therefore are able to stimulate effective killing of HCMV infected cells and have done so in infected, asymptomatic persons. The peptides of this invention are the epitopes which are routinely and successfully presented on the surface of antigen presenting cells in the human host, guaranteeing productive binding to MHC Class I and the elicitation of a cellular immune response to HCMV in human beings.
Truncations of the pp150 protein expressed in vaccinia viruses were screened against pp150-specific T cell clones. These CTL clones were established from HCMV-seropositive volunteers using established methods. (Walter et al., N. Eng. J. Med. 333(16):1038-1044 (1995); McLaughlin-Taylor et al., J. Med. Virol. 43:103-110 (1994); Yee et al., J. Immunol. 157(9):4074-4086 (1996); Diamond et al., Blood, 90:1751-1767 (1997); LaRosa et al., Blood 97:1776-1786 (2001)). Recombinant vaccinia viruses having successive amino and carboxyl terminal deletions of approximately 100-200 nucleotides over the entire pp150 gene were tested for the ability to sensitize cells for killing by the pp150-specific T cells. Progressively smaller truncated peptides covering the length of the identified sequence were tested until a narrow area of the protein was identified as containing the peptide which mediated the cytotoxic T cell response of that clone. When a peptide no longer than 100 amino acids was identified, a series of overlapping peptides covering the length of the identified sequence were synthesized for further analysis. Using these methods, a scan of the 100 amino acid sequence using 15mer peptides overlapping by three amino acids required a total of 20 peptides.
For the test, autologous and HLA mismatched (control) lymphocyte cell lines were sensitized with the scanning peptides at a concentration of 50 xcexcM for 1-2 hours, and washed. The relevant CTL then were incubated with chromated EBVLCL (Epstein-Barr virus transformed lymphocyte cell lines) sensitized with peptide, and a standard chromium release assay was performed. The sensitivity of lysis was determined, and any positive peptide was further truncated, both at the amino and carboxyl termini, until a minimal cytotoxic epitope that corresponds to the HLA allele of that T cell clone was defined. Table I provides peptide epitopes which are naturally processed from pp150 by persons having the indicated HLA allele.
The vaccine epitopes, regardless of primary structure, may be injected s.c. into the forearm or other body location in a standard formulation buffer (PBS/10% DMSO or higher concentration/0.01% triflouroacetic acid or other acid or alcohol of the same or different concentration) once. Vaccines may be administered in PBS or any other pharmaceutically compatible vehicle. Three to six weeks later, a booster injection of the same material may be administered. Multiple booster injections spaced three to six weeks apart can be subsequently administered, if necessary.
Vaccines can be administered to a patient or at-risk individual, or to the donor of a bone marrow transplant, who is either positive or negative for the virus. Illustrative examples of vaccine peptides include:
wherein X is cyclohexylalanine or phenylalanine and xe2x80x9cPamxe2x80x9d is palmitic acid. The three-A or alternative structural spacer (underlined) may be interchanged among vaccine peptides. The format of the peptides shown above can be described (from the amino terminus) as: lipid-KSSxe2x80x94HTL epitope (italics)xe2x80x94amino acid spacer (underlined)xe2x80x94CTL epitope. The positions of the CTL and HTL epitopes may be interchanged. The CTL epitope (or a functional sequence variant thereof) may be further modified by adding a leader sequence and/or the amino acids KDEL may be appended to the carboxyl terminus to assist retention and targeting into the endoplasmic reticulum as exemplified in SEQ ID NO: 8. Palmitic acid or any suitable lipid may be used, including but not limited to stearic acid, myristic acid, lauric acid, capric acid and decanoic acid. Preferred lipid moieties include palmitic acid. Alternatively, forms of the vaccine without lipids may be used, choosing the appropriate T-helper epitope that causes immunogenicity either with or without accompanying adjuvants. Sequences such as KSS may be included at the amino terminus of unlipidated peptides to aid in solubility. Other vaccine formulations include peptides having the dextro form of the amino acid on the N-terminus. Unlipidated vaccines do not require the KSS linker sequence.
Adjuvants may form part of the vaccine formulation. Adjuvants such as complete or incomplete Freund""s adjuvant, aluminum hydroxide or the like are contemplated, however a preferred adjuvant, particularly for use in humans, is a DNA adjuvant. Single-stranded DNA adjuvants comprising specific sequences including Cytosine-phosphate-Guanosine (CpG) are known in the art and are contemplated for use with this invention. DNA adjuvants lacking these CpG sequences also are useful with the invention. An exemplary DNA adjuvant may comprise a 20mer of nucleotides with 2 CpG motifs, or any DNA oligomer, generally about 20 to about 25 nucleotides long. Increased stability of the sequence may be obtained by substituting phosphate groups in the nucleotide backbone with thioate groups to create a phosphoro-thioate backbone rather than a phosphoro-diester backbone.
Vaccines of this invention also may be formulated as DNA vaccine. Suitable vaccines include recombinant viral vectors, for example pox virus, which express a gene encoding one or more HCMV peptides or analogs of the invention. These vaccines may be constructed according to methods known in the prior art. In summary, these peptides may be administered as a vaccine, alone or combined with other peptide sequences, in the presence or absence of an adjuvant. Alternatively, a minimal CTL epitope from an immunogenic protein that is delivered utilizing a virus or DNA construct may also induce CTL responses which have been shown to be important for virus reduction and elimination.
The peptides of this invention also may be used in immunological methods to detect pp150-reactive CTL in a patient or a sample from a patient. Assays such as chromium release assays as described below or any known assay is suitable. Specific T cell clones which recognize pp150 peptide may be detected using an immunological reagent comprising the peptides according to SEQ ID NO: 1 or 2, for example, tetramer reagents such as those described in Altman et al., Science 274:94-96, 1996 or U.S. Pat. No. 5,734,023, the disclosures of which are hereby incorporated by reference, or dimer reagents such as those described in La Rosa et al., Blood 97(6):1776-1786, 2001 and Greten et al., Proc. Natl. Acad. Sci. USA 95:7568-7573, 1998, the disclosures of which are hereby incorporated by reference.
MHC tetramers generally are known in the art and consist of tetrameric complexes of beta-2 microglobulin, a biotinylated MHC class I molecule conjugated to streptavidin linked to a fluorescent marker, and an antigenic peptide such as, for example, a pp150 peptide or the like. The MHC class I allele and the peptide in combination allow specific recognition of T cells which recognize that peptide antigen in the context of the class I allele. Multiple complexes are often linked together to increase binding, since the affinity of the individual complex is generally low. Using a fluorescently labeled tetramer, specifically binding T cells may be separated using known techniques, such as fluorescent activated cell sorting and the like. Dimeric complexes of the same diagnostic reagents taking advantage of pp150 peptide antigens also may be used.
Those of skill in the art are familiar with the use of such dimer and tetramer reagents and are fully able to construct and use such reagents for use in various diagnostic methods known in the art. As well, those of skill in the art can readily synthesize useful reagents or variants of these reagents.
The following examples are intended to illustrate rather than limit the appended claims.